The Role of Toll-Like Receptor 2 in the Recognition of Aggregatibacter actinomycetemcomitans

Background: Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) is a Gram-negative bacterium present in the oral cavity and is usually associated with localized aggressive periodontitis. Isolated antigens from A. actinomycetemcomitans can activate innate immune cells through Toll-like receptors (TLRs), which are molecules that recognize structural components conserved among microorganisms. In this study, we evaluate the role of TLR2 in the recognition of A. actinomycetemcomitans. Methods: Macrophages and neutrophils from knockout mice with targeted disruption of TLR2 (TLR2(-/-) mice) and wild-type mice were collected and used for the subsequent assays. The production of cytokines and chemokines was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of apoptotic cells was determined by flow cytometry. In addition, the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR2(-/-) mice were examined. Results: The results show that TLR2-deficient mice developed more severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly higher bone loss and inflammatory cell migration to periodontal tissues. The inflammatory cell influx into the peritoneal cavities of TLR2(-/-) mice was three-fold lower than that observed for the littermate controls. A significantly diminished production of the cytokines tumor necrosis factor-alpha and interleukin-1 beta as well as the chemokine CC-ligand-5 in the peritoneal cavities of TLR2(-/-) mice was observed. In addition, a high frequency of apoptotic cells in the inflammatory exudates from TLR2(-/-) mice was observed. Phagocytosis and nitric oxide production was diminished in cells from TLR2(-/-) mice, facilitating the dissemination of the pathogen to the spleen. Conclusion: The results of this study highlight the involvement of TLR2 in recognizing A. actinomycetemcomitans and its essential role in controlling A. actinomycetemcomitans infection. J Periodontot 2009,80:2070-2019.

Use of the DNA Checkerboard hybridization method for detection and quantitation of Candida species in oral microbiota

The DNA Checkerboard method enables the simultaneous identification of distinct microorganisms in a large number of samples and employs up to 45 whole genomic DNA probes to gram-negative and gram-positive bacterial species present in subgingival biofilms. Collectively, they account for 55%-60% of the bacteria in subgingival biofilms. In this study, we present the DNA Checkerboard hybridization as an alternative method for the detection and quantitation of Candida species in oral cavities. Our results reveal that DNA Checkerboard is sensitive enough and constitutes a powerful and appropriate method for detecting and quantifying Candida species found in the oral cavity.

Antimicrobial and cytotoxic activities of medicinal plants of the Brazilian cerrado, using Brazilian cachaca as extractor liquid

Ethnopharmacological importance: Many species of plants in the Brazilian cerrado (savanna) are widely used in ethnomedicine. However, the safety and effectiveness of medicinal plants used in communities with little or no access to manufactured drugs should be evaluated. Aim of the study: Evaluate the antimicrobial and cytotoxic activities of extracts from eight plant species, obtained using Brazilian cachaca as the extractor liquid. Materials and methods: The extracts were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Candida parapsilosis, promastigote forms of Leishmania amazonensis, and poliovirus. In addition, cytotoxic activity was assayed in Vero cells and in human erythrocytes. Results: The plant species Curatella americana, Sclerolobium aureum, and Plathymenia reticulata showed the best activity against yeasts, especially the crude extract of C. americana and its ethyl-acetate fraction. Kielmeyera lathrophyton showed a minimum inhibitory concentration of 250 mu g/ml against S. aureus, and was inactive against Gram-negative bacteria. The extract obtained from Annona coriacea showed the best activity against the promastigote forms of Leishmania amazonensis (IC(50) = 175 mu g/ml). Only C. americana showed potential for antipoliovirus activity. The concentrations of the crude extracts that showed toxicity to VERO cells had CC(50) between 31 and 470 mu g/ml, and the lyophilized Brazilian cachaca showed a CC(50) of 307 mu g/ml. None of the extracts showed toxicity against human erythrocytes. Conclusions: Among the plant species studied. C americana proved to be effective against microorganisms, especially as an antifungal. The results will help in the search for alternative drugs to be used in pharmacotherapy, and will contribute to establish safe and effective use of phytomedicines in the treatment of infectious diseases. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Brazilian red propolis: unreported substances, antioxidant and antimicrobial activities

BACKGROUND: Chloroform, ethyl acetate and methanol extracts of a sample of red propolis from the state of Alagoas (northeast Brazil) were analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry. Antimicrobial and antioxidant activities were also obtained. RESULTS: The propolis sample contained low content of narigenin-8-C-hexoside, this being the first report of a C-glycoside in propolis. The main constituent found was characterized as 3,4,2`,3`-tetrahydroxychalcone. Other important constituents were the chalcone isoliquiritigenin, the isoflavans (3S)-vestitol, (3S)-7-O-methylvestitol, the pterocarpan medicarpin, the phenylpropenes trans-anethol, methyl eugenol, elimicin, methoxyeugenol and cis-asarone, and the triterpenic alcohols lupeol and alpha- and beta- amyrins. The methanol extract exhibited high antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl and beta-carotene/linoleic acid assay methods, and antimicrobial activity toward Gram-positive and Gram-negative bacteria. CONCLUSION: Structures are suggested for new substances never before seen in any kind of propolis. This is the first report of 3,4,2`,3`-tetrahydroxychalcone and a flavone C-glycoside in a propolis sample. (C) 2011 Society of Chemical Industry

Melatonin inhibits LPS-induced NO production in rat endothelial cells

Endothelial cells produce NO by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NOS (iNOS). We have previously shown that melatonin, in the nanomolar range, inhibits activation of constitutive NOS, and in the present paper, we evaluated whether it could interfere with the expression of iNOS, which is activated by lipopolysaccharide (LPS), a major component of gram-negative bacteria cell walls. Primary cultures of rat endothelial cells were loaded with fluorescent probe for NO detection. Nuclear factor kappa B (NF-kappa B) translocation in endothelial cells elicited by LPS was measured by electromobility shift assay, and the vasodilation of aortic rings was accessed by recording isometric contraction. Melatonin in a micromolar but not in a nanomolar range inhibits the NO production induced by LPS. This effect is not dependent on the activation of G protein-coupled melatonin receptors. The nuclear NF-kappa B translocation is a process necessary for iNOS transcription, and melatonin also inhibits its translocation. LPS induced vasodilation only in endothelium-intact aortic rings, and melatonin (10 mu m) inhibits the vasodilation. Here, we show that concentrations compatible with nocturnal melatonin surge (nm) did not interfere with the activity of iNOS. Considering that micromolar melatonin concentrations could be locally achieved through production by activated immune competent cells, extra-pineal melatonin could have a protective effect against tissue injury. We propose that melatonin blocked the LPS-induced vasodilation by inhibiting the NF-kappa B pathway. Finally, we propose that the effect of melatonin on vascular reactivity is one of the mechanisms that underlies the protective effect of this indolamine against LPS.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway

Nuclear factor-kappa B (NFKB), a pivotal player in inflammatory responses, is constitutively expressed in the pineal gland. Corticosterone inhibits pineal NFKB leading to an enhancement of melatonin production, while tumor necrosis factor (TNF) leads to inhibition of Aa-nat transcription and the production of N-acetylserotonin in cultured glands. The reduction in nocturnal melatonin surge favors the mounting of the inflammatory response. Despite these data, there is no clear evidence of the ability of the pineal gland to recognize molecules that signal infection. This study investigated whether the rat pineal gland expresses receptors for lipopolysaccharide (LPS), the endotoxin from the membranes of Gram-negative bacteria, and to establish the mechanism of action of LPS. Here, we show that pineal glands possess both CD14 and toll-like receptor 4 (TLR4), membrane proteins that bind LPS and trigger the NFKB pathway. LPS induced the nuclear translocation of p50/p50 and p50/RELA dimers and the synthesis of TNF. The maximal expression of TNF in cultured glands coincides with an increase in the expression of TNF receptor 1 (TNFR1) in isolated pinealocytes. In addition, LPS inhibited the synthesis of N-acetylserotonin and melatonin. Therefore, the pineal gland transduces Gram-negative endotoxin stimulation by producing TNF and inhibiting melatonin synthesis. Here, we provide evidence to reinforce the idea of an immune-pineal axis, showing that the pineal gland is a constitutive player in the innate immune response.

Insulin suppresses LPS-induced iNOS and COX-2 expression and NF-kappa B activation in alveolar macrophages

The development of septic shock is a common and frequently lethal consequence of gram-negative infection. Mediators released by lung macrophages activated by bacterial products such as lipopolysaccharide (LPS) contribute to shock symptoms. We have shown that insulin downregulates LPS-induced TNF production by alveolar macrophages (AMs). In the present study, we investigated the effect of insulin on the LPS-induced production of nitric oxide (NO) and prostaglandin (PG)-E(2), on the expression of inducible nitric oxide synthase ( iNOS) and cyclooxygenase (COX)-2, and on nuclear factor kappa B (NF-kappa B) activation in AMs. Resident AMs from male Wistar rats were stimulated with LPS (100 ng/mL) for 30 minutes. Insulin (1 mU/mL) was added 10 min before LPS. Enzymes expression, NF-kappa B p65 activation and inhibitor of kappa B (I-kappa B) a phosphorylation were assessed by immunobloting; NO by Griess reaction and PGE(2) by enzyme immunoassay (EIA). LPS induced in AMs the expression of iNOS and COX-2 proteins and production of NO and PGE(2), and, in parallel, NF-kappa B p65 activation and cytoplasmic I-kappa B alpha phosphorylation. Administration of insulin before LPS suppressed the expression of iNOS and COX-2, of NO and PGE(2) production and Nuclear NF-kappa B p65 activation. Insulin also prevented cytoplasmic I-kappa Ba phosphorylation. These results show that in AMs stimulated by LPS, insulin prevents nuclear translocation of NF-kappa B, possibly by blocking I-kappa Ba degradation, and supresses the production of NO and PGE(2), two molecules that contribute to septic shock. Copyright (C) 2008 S. Karger AG, Basel.

Insulin inhibits LPS-induced signaling pathways in alveolar macrophages

The systemic inflammatory response syndrome ( SIRS) is triggered by lipopolysaccharide (LPS) from Gram-negative bacteria. Insulin was shown to have a protective role in SIRS related to sepsis. Lungs are particularly affected in this condition and provide a second wave of mediators/cytokines which amplifies SIRS. The aim of the present study was to investigate the effect of insulin on the signaling pathways elicited by LPS in alveolar macrophages (AMs) and its consequence in cellular response to LPS measured as production of tumor necrosis factor (TNF). To this purpose, resident AMs from male Wistar rats were obtained by lung lavage and stimulated by LPS ( 100 ng/mL). Insulin ( 1 mU/mL) was added 10 min before LPS. Activation ( phosphorylation) of signaling molecules by LPS was analyzed by western blot, 30 min after LPS stimulation. TNF was measured in the AMs culture supernatants by bioassay using L-929 tumor cells. Relative to controls, LPS induced a significant increase in the activation of ERK (3.6-fold), p38 (4.4-fold), Tyr-326 Akt (4.7-fold), Ser-473 Akt (6.9-fold), PKCa (4.7-fold) and PKCd (2.3-fold). Treatment of AMs with insulin before LPS stimulation, significantly reduced the activation of ERK (54%), p38 (48%), Tyr-326 Akt (64%), Ser-473 Akt (41%), PKCa (62%) and PKCd (39%). LPS induced TNF production in AMs which was also inhibited by insulin (60%). These results show that insulin down-regulates MAPK, PI3K and PKCs and inhibits a downstream effect of LPS, TNF production, in rat AMs stimulated with LPS and suggest that the protective effect of insulin in sepsis could be through modulation of signal transduction pathways elicited by LPS in lung macrophages. Copyright (c) 2008 S. Karger AG, Basel.

Inhibition of interferon-gamma-induced nitric oxide production in endotoxin-activated macrophages by cytolethal distending toxin

Introduction: Cytolethal distending toxin (CDT) is a DNA-targeting agent produced by certain pathogenic gram-negative bacteria such as the periodontopathogenic organism Aggregatibacter actinomycetemcomitans. CDT targets lymphocytes and other cells causing cell cycle arrest and apoptosis, impairing the host immune response and contributing to the persistence of infections caused by this microorganism. In this study we explored the effects of CDT on the innate immune response, by investigating how it affects production of nitric oxide (NO) by macrophages. Methods: Murine peritoneal macrophages were stimulated with Escherichia coli sonicates and NO production was measured in the presence or not of active CDT. Results: We observed that CDT promptly and significantly inhibited NO production by inducible nitric oxide synthase (iNOS) in a dose-dependent manner. This inhibition is directed towards interferon-gamma-dependent pathways and is not mediated by either interleukin-4 or interleukin-10. Conclusion: This mechanism may constitute an important aspect of the immunosuppression mediated by CDT and may have potential clinical implications in A. actinomycetemcomitans infections.

Screening for endophytic nitrogen-fixing bacteria in Brazilian sugar cane varieties used in organic farming and description of Stenotrophomonas pavanii sp. nov.

A Gram-negative, rod-shaped, non-spore-forming and nitrogen-fixing bacterium, designated ICB 89(T), was isolated from stems of a Brazilian sugar cane variety widely used in organic farming. 16S rRNA gene sequence analysis revealed that strain ICB 89(T) belonged to the genus Stenotrophomonas and was most closely related to Stenotrophomonas maltophilia LMG 958(T), Stenotrophomonas rhizophila LMG 22075(T), Stenotrophomonas nitritireducens L2(T), [Pseudomonas] geniculata ATCC 19374(T), [Pseudomonas] hibiscicola ATCC 19867(T) and [Pseudomonas] beteli ATCC 19861(T). DNA-DNA hybridization together with chemotaxonomic data and biochemical characteristics allowed the differentiation of strain ICB 89(T) from its nearest phylogenetic neighbours. Therefore, strain ICB 89(T) represents a novel species, for which the name Stenotrophomonas pavanii sp. nov. is proposed. The type strain is ICB 89(T) (=CBMAI 564(T) =LMG 25348(T)).

PHB Biosynthesis in Catabolite Repression Mutant of Burkholderia sacchari

Due to the effect of catabolite repression, sugar mixtures cannot be metabolized in a rapid and efficient way implicating in lower productivity in bioprocesses using lignocellulosic hydrolysates. In gram-negative bacteria, this mechanism is mediated by the phosphotransferase system (PTS), which concomitantly internalizes and phosphorylates sugars. In this study, we isolated a UV mutant of Burkholderia sacchari, called LFM828, which transports hexoses and pentoses by a non-PTS uptake system. This mutant presented released glucose catabolite repression over the pentoses. In mixtures of glucose, xylose, and arabinose, specific growth rates and the specific sugar consumption rates were, respectively, 10 and 23% higher in LFM828, resulting in a reduced time to exhaust all sugars in the medium. However, in polyhydroxybutyrate (PHB) biosynthesis experiments it was necessary the supplementation of yeast extract to maintain higher values of growth rate and sugar consumption rate. The deficient growth in mineral medium was partially recovered by replacing the ammonium nitrogen source by glutamate. It was demonstrated that the ammonium metabolism is not defective in LFM828, differently from ammonium, glutamate can also be used as carbon and energy allowing an improvement on the carbohydrates utilization for PHB production in LFM828. In contrast, higher rates of ammonia consumption and CO(2) production in LFM828 indicate altered fluxes through the central metabolism in LFM828 and the parental. In conclusion, PTS plays an important role in cell physiology and the elimination of its components has a significant impact on catabolite repression, carbon flux distribution, and PHB biosynthesis in B. sacchari.

Screening of bacteria to produce polyhydroxyalkanoates from xylose

Although xylose is a major constituent of lignocellulosic feedstock and the second most abundant sugar in nature, only 22% of 3,152 screened bacterial isolates showed significant growth in xylose in 24 h. Of those 684, only 24% accumulated polyhydroxyalkanoates after 72 h. A mangrove isolate, identified as Bacillus sp. MA3.3, yielded the best results in literature thus far for Gram-positive strains in experiments with glucose and xylose as the sole carbon source. When glucose or xylose were supplied, poly-3-hydroxybutyrate (PHB) contents of cell dry weight were, respectively, 62 and 64%, PHB yield 0.25 and 0.24 g g(-1) and PHB productivity (P(PHB)) 0.10 and 0.06 g l(-1) h(-1). This 40% P(PHB) difference may be related to the theoretical ATP production per 3-hydroxybutyrate (3HB) monomer calculated as 3 mol mol(-1) for xylose, less than half of the ATP/3HB produced from glucose (7 mol mol(-1)). In PHB production using sugar mixtures, all parameters were strongly reduced due to carbon catabolite repression. PHB production using Gram-positive strains is particularly interesting for medical applications because these bacteria do not produce lipopolysaccharide endotoxins which can induce immunogenic reactions. Moreover, the combination of inexpensive substrates and products of more value may lead to the economical sustainability of industrial PHB production.

Disruption of the 2-methylcitric acid cycle and evaluation of poly-3-hydroxybutyrate-co-3-hydroxyvalerate biosynthesis suggest alternate catabolic pathways of propionate in Burkholderia sacchari

The objective of the present work was to evaluate the relevance of the 2-methylcitric acid cycle (2MCC) to the catabolism of propionate in Burkholderia sacchari. Two B. sacchari mutants unable to grow on propionate were obtained: one disrupted in acnM, and the other in acnM and prpC deleted. An operative 2MCC significantly reduces the bacterial ability to incorporate 3-hydroxyvalerate (3HV) into a biodegradable copolyester accumulated from carbohydrates plus propionate. The efficiency of the mutants in converting propionate to 3HV units (Y(3HV/prp)) increased from 0.09 g.g(-1) to 0.81-0.96 g.g(-1), indicating that acnM and prpC are both essential for growth on propionate. None of the mutations resulted in achievement of the maximum theoretical Y(3HV/prp) (1.35 g.g(-1)). When increasing concentrations of propionate were supplied, decreasing values of Y(3HV/prp) were observed. The results obtained corroborate the hypothesis of the presence of other propionate catabolic pathways in B. sacchari. The 2MCC would be the more operative pathway, but a second pathway, which remains to be elucidated, would assume more importance under propionate concentrations of 1 g.L(-1) or higher. The efficiency in converting propionate to 3HV units can be improved by decreasing the propionate concentrations, owing to the role of the 2MCC.

Violacein Extracted from Chromobacterium violaceum Inhibits Plasmodium Growth In Vitro and In Vivo

Violacein is a violet pigment extracted from the gram-negative bacterium Chromobacterium violaceum. It presents bactericidal, tumoricidal, trypanocidal, and antileishmanial activities. We show that micromolar concentrations efficiently killed chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro; inhibited parasitemia in vivo, even after parasite establishment; and protected Plasmodium chabaudi chabaudi-infected mice from a lethal challenge.

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira. (C) 2008 Elsevier Ltd. All rights reserved.